Introduction: Diffuse Alveolar Damage (DAD) is the dominant histological manifestation of severe COVID-19 in the lungs. Understanding tissue immune responses has been limited by failure to examine distinct injury patterns that reflect temporal phases of DAD evolution.

Aims & Objectives: To determine whether there are unique cellular signatures across temporal phases of COVID-19 DAD.

Methods: Lung tissue was obtained from patients who died from COVID-19. Pathologist-selected 500µm2 regions of interest (ROIs) were classified as exudative DAD (EDAD) or organising (ODAD) by light microscopy. ROIs free of DAD from SARS-CoV-2-negative donors and positive patients served as controls. ROIs were stained with 40 immune, functional and stromal markers and ablated using imaging mass cytometry. Segmented cells were classified by FlowSOM clustering. Cell populations and spatial relationships were compared between ROI classes to look for characterising cellular signatures.

Results: Forty patients (80% male, age 22-98), 345 ROIs and >900k single cells were analysed. Mononuclear phagocytes (MnPs) increased in EDAD from control (median 12.7 v 8.6% total cells, p<0.0001) and ODAD from EDAD (18.9 v 12.7%, p<0.0001). T lymphocytes increased in EDAD from control (median 7.0 v 4.1%, p<0.0001) and ODAD from EDAD (8.6 v 7.0%, p<0.004). B lymphocytes also increased in EDAD from control (median 1.8 v 0.8%, p<0.0001) and ODAD from EDAD (2.7 vs 1.8%, p<0.0029). No differences were observed in neutrophil proportions. Spatial analysis reveals key cellular interactions are established prior to overt pathology.

Conclusions: Sustained increases in MnPs and lymphocytes define the evolutionary signature of COVID-19 DAD.